Enzymatic activity based-detection

ABSTRACT

Disclosed herein are methods and kits which are useful for detecting presence of an enzyme in a test sample based upon the intrinsic enzymatic activity of such test sample. The present invention provides the ability to evaluate cell culture conditions and optimize the desired glycoform content of recombinantly prepared enzymes.

RELATED APPLICATION(S)

This application is a continuation of U.S. application Ser. No.13/513,350, filed Jul. 31, 2012, now U.S. Pat. No. 8,778,622, which wasthe National Stage of International Application No. PCT/US2010/058454,filed Nov. 30, 2010, which claims the benefit of U.S. ProvisionalApplication No. 61/265,620, filed Dec. 1, 2009, the entire teachings ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Glycoproteins and glycoenzymes are proteins that contain apost-translational modification wherein oligosaccharide chains (known asglycans) are covalently attached to the protein's or enzyme'spolypeptide side chains. This process, which is known as glycosylation,is one of the most abundant protein post-translational modifications. Itis estimated that more than half of all cellular and secretory proteinsare glycosylated. (Apweiler et al., 1999, Biochim. Biophys. Acta 1473:4-8). Although mammalian glycoprotein oligosaccharides, for example, areconstructed from a limited number of monosaccharides, their structuraldiversity is vast due to complex branching patterns. Glycoproteins,therefore, represent a diverse group of modifications, and variants ofglycoproteins or glycoenzymes (which are known as glycoforms) can impactprotein or enzyme activity or function. The ability to evaluate anddistinguish specific glycan structures during the preparation ofrecombinant enzymes can accordingly provide valuable informationrelating to recombinant enzyme development and further optimization ofthe desired glycoform content of such recombinant enzymes.

Conventional techniques which are routinely employed for glycoproteinand glycoenzyme analysis include mass spectrometry, lectin affinitychromatography and western blotting. Although these conventional methodsof analysis are generally accurate, they are time consuming, requirepurification of the protein, and some, such as mass spectrometry,require specific expertise and are technically challenging. (Wang etal., 2006, Glycobiol. Epub.; Qiu et al., 2005, Anal, Chem. 77:2802-2809;Qiu et al., 2005, Anal Chem. 77:7225-7231; Novotny et al., 2005, J. Sep.Sci. 28:1956-1968). Accordingly, these issues make the routine use ofsuch technologies impractical for high-throughput monitoring of enzymeglycosylation, especially during process development and manufacturing.Such technologies may also present challenges to a typical researchlaboratory attempting to study the impact of glycosylation on thebiological properties of proteins and enzymes.

Traditionally, to provide a quantitative assessment of the glycanstructure of a glycoprotein, lectin array platforms required the use ofeither a reliable glycoprotein specific antibody or direct conjugationof a fluorescent dye to the glycoprotein. These antibody-based detectionstrategies are limited by the fact that antibody recognition of a givenglycoprotein or glycoenzyme may be blocked or reduced depending on thetype of glycan structure linked to the protein or enzyme, therebyallowing recognition of only a subset of the total glycoprotein pool andnot the range of potential glycan structures. Antibody-based recognitionmay also require multiple binding and wash steps, which can add time andcomplexity to an analysis. While these problems can be circumventedusing direct labeling of the glycoprotein, direct labeling remainslimited to pure preparations of material, since the labeling techniquesdo not discriminate among proteins. Accordingly, direct labeling cannotbe used for “dirty” or in-process samples. The utility of currentlyavailable methods for glycan analysis may be further limited becauselarge quantities of highly purified materials may not readily beavailable from in-process test samples. Furthermore, purified materialmay only represent a subset of the initial glycoform population becausethe purification process is typically selective for certain glycanstructures.

The identification and characterization of protein and enzyme glycoformsis essential in the development of recombinant proteins and enzymes. Forexample, glycosylation of recombinantly-prepared enzymes must frequentlybe controlled during production to maintain the efficacy and safety ofsuch recombinant enzymes, and cell culture conditions can affect thecarbohydrate structures of glycoproteins. Further understanding of cellculture conditions that can impact the carbohydrate structures ofrecombinantly-prepared proteins or enzymes is also important for thedevelopment of an effective and robust recombinant production process.

Improved methods and compositions are needed for the rapid, direct andsystematic identification and evaluation of the glycan structures of agiven protein or enzyme and their variant glycoforms. High throughputmethods and compositions that are capable of efficiently assessing anddistinguishing among a diverse range of glycosylation states orglycoforms would provide valuable information for drug discovery anddisease therapeutics, provide valuable tools regarding ongoing research,and facilitate the optimization of recombinant production processes.

SUMMARY OF THE INVENTION

The present invention provides novel methods, assays and compositionsfor the accurate and rapid identification and/or detection of variousglycoforms of enzymes. In particular, the present invention relies uponthe intrinsic activity of the enzyme of interest to detect such enzyme'spresence in a test sample. The methods, assays and compositionsdisclosed herein also provide novel strategies for analyzing thedifferent glycoforms of unpurified proteins or enzymes in cell cultureharvest test samples. Furthermore, the present invention provides theability to detect and distinguish among different glycoforms orglycovariants of an enzyme in upstream harvest test samples, therebyfacilitating the optimization of cell culture conditions that affect theviable glycoform content of recombinantly-prepared enzymes. The methodsand kits of the present invention are advantageously capable ofdetermining the presence of glycosylated enzymes in a test sampleirrespective of the presence of additional cellular proteins, biologicalmaterials or other contaminants which may be present in that testsample.

Disclosed herein are methods for detecting the presence of an enzyme(e.g., a recombinantly prepared enzyme) in a test sample, such methodscomprising contacting the test sample with at least one capture agent(e.g., a lectin) under conditions appropriate for binding ofglycosylated enzyme in the test sample to the capture agent, whereinupon binding of glycosylated enzyme to capture agent a complex is formedwhich is referred to herein as a “bound enzyme.” Such methods alsocontemplate separation of the test sample from the bound enzyme producedby the previous step (e.g., using routine means such as washing)followed by detection of the intrinsic enzymatic activity of the boundenzyme. The presence of intrinsic enzymatic activity is indicative ofthe presence of enzyme in the test sample.

Also disclosed are methods for detecting the presence of an enzyme(e.g., a recombinantly prepared enzyme) in a test sample, wherein suchmethods comprise the steps of contacting a test sample with at least onecapture agent (e.g., a lectin) under conditions appropriate for bindingof the glycosylated enzyme, and thereby forming a bound enzyme whenglycosylated enzyme is present. The methods of the present inventionalso contemplate separating the bound enzyme from the test sample andcontacting the bound enzyme with at least one substrate. In accordancewith the present invention, the presence of intrinsic enzymatic activityof such bound enzyme is indicative of the presence of the glycosylatedenzyme of interest in the test sample. Conversely, the absence ofintrinsic enzymatic activity is indicative of the absence of theglycosylated enzyme of interest in the test sample. The methodsdisclosed herein provide the ability to optimize the desired glycoformcontent of one or more recombinant enzymes during recombinantpreparation.

In one embodiment, the methods of the present invention further comprisethe step of fixing a capture agent (e.g., one or more lectins) onto asolid support (e.g., a microtiter plate or one or more populations ofbeads). In one embodiment, such solid support may comprise or be coatedwith avidin, streptavidin or a metal chelator such as a nickel chelate.If such solid support comprises avidin or streptavidin, the use ofderivatized lectins (e.g., biotinylated lectins) are preferred. If suchsolid support comprises a nickel chelate, the use of six consecutivehistidine residues (6His) as an affinity tag is preferred. For example,a capture agent may be a fusion protein which includes one or morehistidine (HIS) residues (e.g., at least one, at least two, at leastthree, at least four, at least five, at least six, at least eight, atleast ten, at least twelve, at least twenty, at least twenty five ormore HIS residues) at either the N- or C-terminus as an affinity tag tofacilitate fixing of that capture agent (i.e., the fusion protein) to asolid support.

In one embodiment of the present invention the capture agent comprisesone or more lectins. The lectins contemplated by the methods, assays andkits of the present invention include, for example, concanavalin A,wheat germ agglutinin, Jacalin, lentil lectin, peanut lectin, lensculinaris agglutinin, Griffonia (Bandeiraea) simplicifolia lectin II,Aleuria aurantia lectin, hippeastrum hybrid lectin, sambucus nigralectin, maackia amurensis lectin II, ulex europaeus agglutinin I, lotustetragonolobus lectin, galanthus nivalis lectin, euonymus europaeuslectin, ricinus communis agglutinin I, and any combinations thereof.

In another embodiment of the present invention the capture agentcomprises a receptor, or a binding fragment thereof, known todemonstrate affinity for or otherwise bind to one or more particularglycoforms of an enzyme. For example, mannose-6-phosphate (M6P) bindsthe mannose-6-phophate receptor (M6PR), and in one embodiment arecombinant fusion protein comprising the M6PR or a binding domainthereof (e.g., M6PR domain 9) may serve as the capture agent. In oneembodiment, the recombinant fusion protein capture agent may alsocomprise one or more histidine residues (e.g., 6His) to facilitatepurification, capture and/or fixing of the capture agent to a solidsupport. In one embodiment of the present invention, the capture agentcomprises the fusion protein M6PR(D9)6His.

Also disclosed is a method of determining the intrinsic enzymaticactivity of the bound enzyme by contacting such bound enzyme with asubstrate, for example, a substrate which has known reactivity with theenzyme suspected of being present in the test sample. In accordance withthe methods of the present invention, the presence of intrinsicenzymatic activity is indicative of the presence of enzyme in the testsample. Alternatively, the absence of intrinsic enzymatic activity maybe indicative of the absence of such enzyme in the test sample.

In one embodiment, the methods, assays and kits of the present inventioncontemplate determining intrinsic enzymatic activity by contacting boundenzyme with a substrate which is known to predictably react with theenzyme of interest. For example, if the enzyme is agalsidase alfa theselected substrate may be 4-nitrophenyl-α-D-galaetopyranoside, if theenzyme is galactocerebrosidase the selected substrate may be4-nitrophenyl-β-D-galactopyranoside, and if the enzyme is aryl sulfataseA the selected substrate may be p-nitrocatechol sulfate. The presence orabsence of intrinsic enzymatic activity of the bound enzyme may bedetermined by means which are known to those of ordinary skill in theart. In one embodiment a quantitative assessment of the depletion ofsubstrate and/or the conversion of substrate to product may beindicative of intrinsic enzymatic activity. For example, in oneembodiment substrate depletion of about 5%, 10%, 20%, 30%, 40%, 50% ormore, or preferably about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,99% or more relative to the amount of substrate introduced may beindicative of intrinsic enzymatic activity. Alternatively, followingcontacting an enzyme with a substrate, a relative increase in theformation of a product, or the conversion of substrate to product, ineach case of about 5%, 10%, 20%, 30%, 40%, 50% or more, or preferablyabout 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100% or more,may be indicative of intrinsic enzymatic activity. Substratescontemplated by the present invention include, for example,4-nitrophenyl-α-D-galactopyranoside, 4-nitrophenyl-β-D-galactopyranosideand para-nitrocatechol sulfate.

Also disclosed herein are kits which are useful for detecting thepresence of glycosylated enzymes (e.g., a recombinantly preparedglycosylated enzyme) in a test sample. Such kits comprise at least onecapture agent (e.g., a lectin) capable of binding a glycosylated enzyme,and at least one substrate which is reactive with such glycosylatedenzyme. In one embodiment the kits of the present invention comprise asolid support (e.g., a multiple well microtiter plate), onto which maybe fixed a capture agent (e.g., the lectin sambucus nigra agglutinin).

In one embodiment, the kits of the present invention comprise a captureagent which is known to bind or demonstrate affinity for the targetedglycoform of the enzyme of interest (e.g., the M6PR(D9)6His fusionprotein), and a substrate which is known to react with such enzyme. Inone embodiment, such kits may also comprise a means of separating orremoving excess test sample from the solid support, for example bywashing, or other routine means available to one of ordinary skill inthe art.

Also contemplated are kits which are capable of identifying multipleglycosylated enzymes and multiple glycoforms of those enzymes in thesame test sample. For example, the kits of the present invention maycomprise multiple capture agents (e.g., lectins) fixed onto one or moresolid supports (e.g., populations of inert beads), and thus provide thecapability of binding to multiple glycoforms of one or more enzymes inthe same test sample. The kits of the present invention may alsocomprise one or more substrates (each of which correspond to aparticular enzyme whose presence is suspected in a test sample) todetermine such enzymes' intrinsic enzymatic activities. Preferably, theselected substrate is known to predictably bind to, or react with, theenzyme of interest. For example, if the enzyme is agalsidase alfa theselected substrate may be 4-nitrophenyl-α-D-galactopyranoside, if theenzyme is galactocerebrosidase the selected substrate may be4-nitrophenyl-β-D-galactopyranoside, and if the enzyme is aryl sulfataseA the selected substrate may be p-nitrocatechol sulfate. Based upon thebinding specificity or reactivity of the test sample with the substrate,one having ordinary skill in the art may use routine means to assess thepresence or absence of intrinsic enzymatic activity (e.g., byquantitatively determining substrate depletion and/or the conversion ofsubstrate to product).

The above discussed and many other features and attendant advantages ofthe present invention will become better understood by reference to thefollowing detailed description of the invention when taken inconjunction with the accompanying examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates one embodiment of the present inventionin which biotinylated lectins are bound to streptavidin-coated plates,to which are added test sample materials containing differentpreparations of a given glycosylated enzyme which are allowed to bind.Unbound test sample materials are then removed by a wash step, andspecific detection of the bound enzyme is performed by the addition ofthe appropriate substrate and assay conditions.

FIG. 2 illustrates binding of agalsidase alfa to immobilized wheat germagglutinin (WGA) and concanavalin A (ConA) as determined by measuringthe enzymatic activity of agalsidase alfa (Replagal®) based on thesubstrate 4-nitrophenyl-α-D-galactopyranoside.

FIG. 3 illustrates binding of galactocerebrosidase (GalC) to immobilizedwheat germ agglutinin (WGA), concanavalin A (ConA), and Sambucus nigralectin (SNA) as determined by measuring enzymatic activity of GalC usingthe substrate 4-nitrophenyl-β-D-galactopyranoside.

FIG. 4 illustrates binding of galactocerebrosidase (GalC) treated withincreasing concentrations of sialidase to immobilized Sambucus nigralectin (SNA), as determined by measuring enzymatic activity of GalCusing the substrate 4-nitrophenyl-β-D-galactopyranoside.

FIG. 5 illustrates linkage-specific binding of purified aryl sulfatase A(ARSA) containing sialic acid in either α-2, 6 or α-2, 3 linkages toSambucus nigra lectin (SNA), as determined by measuring enzymaticactivity of ARSA using the substrate p-nitrocatechol sulfate.

FIG. 6 illustrates binding of galactocerebrosidase (GalC) cell culturefrom different harvest test samples to Sambucus nigra lectin (SNA), asdetermined by measuring enzymatic activity of GalC using the substrate4-nitrophenyl-β-D-galactopyranoside.

FIG. 7 schematically illustrates one embodiment of the present inventionin which the M6PR(D9)6His fusion protein is bound to a nickelchelate-coated 96-well plate, to, which are added test samplescontaining different preparations of a given glycosylated enzyme whichare allowed to bind. Unbound test sample material is then removed by awash step, and specific detection of the bound enzyme is performed bythe addition of the appropriate substrate and assay conditions.

FIG. 8 illustrates detection differences in the amount of aryl sulfataseA (ARSA) associated M6P using ARSA lots with known amounts of M6P.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In addition, the materials,methods, and examples are illustrative only and are not intended to belimiting.

Disclosed herein are high throughput methods, assays, kits andcompositions for screening complex test samples for the presence ofglycosylated enzymes of interest or for determining changes inglycosylation of such enzymes. Also disclosed herein are methods andkits which are capable of detecting the intrinsic activity of an enzymein a test sample as a means of determining the presence of that enzymein the test sample. For example, in one aspect the present inventionrelies upon the intrinsic enzymatic activity of an enzyme to detect itspresence in a test sample. This is in contrast to antibody-baseddetection schemes which can be negatively influenced by changes inglycosylation, for example by hindrance of antibody recognition of theenzyme. The invention provides the ability to study cell cultureconditions and optimize the desired glycoform content of biologicalsamples, and in particular of recombinantly prepared enzymes.

In the context of the present invention, the term “glycan” refers to thecarbohydrate portion of a glycoprotein or glycoenzyme. Generally,glycans tend to be oligosaccharides or polysaccharides. The terms“glycoform” and “glycovariant” refer to an isoform of a protein orenzyme with a unique primary, secondary, tertiary, and/or quaternarystructure based upon the number and/or structure of the glycans attachedto such protein or enzyme. It is often the case that a singleglycoprotein may have over a thousand different glycoforms orglycovariants, all of which are based on differences in the glycanportion or glycosylation pattern of the glycoprotein. The term“glycosylation” refers to the process or result of adding saccharides toproteins and thus forming “glycoproteins”. Glycosylation includes bothN-linked glycosylation to the amide nitrogen of asparagine side chains,and O-linked glycosylation to the hydroxy oxygen of serine and threonineside chains.

The term “test sample” is used in its broadest sense and means anypreparation, preferably obtained from biological media or materials,including biologically or recombinantly derived media which may contain,among other things, naturally occurring or recombinantly preparedpeptides, polypeptides or proteins, enzymes, lipid or carbohydratemolecules, or glycosylated proteins or enzymes, or other samplesobtained from a recombinant media, including any fractions thereof. Thetest samples contemplated by the present invention are preferablyobtained from in-process or “dirty” biological systems, for example,those obtained during the preparation of a recombinant enzyme.

As used herein, the term “solid support” refers to any material thatprovides a solid or semi-solid structure with or upon which anothermaterial can be attached or fixed. Such materials include smoothsupports (e.g., metal, glass, plastic, silicon, and ceramic surfaces) aswell as textured and porous materials. Such materials also include, butare not limited to, gels, rubbers, polymers, dendrimers and othernon-rigid materials. Solid supports need not be flat. Supports includeany type of shape including spherical shapes (e.g., beads) and mayinclude multiple well microtiter plates, and may optionally be coatedwith proteins, resins or other similar reagents, such as for example,avidin, streptavidin, metal ions or chelates (e.g., a nickel chelate).

The present invention contemplates the use of one or more capture agentsto facilitate capture, immobilization or fixing of the glycosylatedenzyme of interest (e.g., capture or fixing of one or more glycosylatedenzymes onto a solid support). As used herein, the phrase “captureagent” refers to a compound or a material which demonstrates affinityfor or is capable of conjugating or associating with one or morespecific saccharide or carbohydrate moieties. Preferably the selectedcapture agent demonstrates specific or discriminatory affinity for onesaccharide moiety, such that the capture agent will only bind oneparticular glycoform of a given enzyme. In a preferred embodiment of thepresent invention, the capture agent is selected based upon its specificaffinity to one or more glycan structures. When contacted with such aglycan structure or glycosylated enzyme in accordance with the presentinvention the capture agent will form a complex referred to herein as a“bound enzyme.”

In one embodiment of the present invention, the capture agent is alectin. Lectins represent a family of saccharide-recognizing proteinswhich are classified based upon the specificity of the mono-saccharidegroups for which they exhibit the highest affinities. Lectins arenon-enzymatic and non-immune in nature and are capable of binding to thesaccharide moiety of a glycoprotein or glycoenzyme. As used herein, theterm “lectin” refers to a non-antibody compound which binds to aspecific carbohydrate structure or target, such as for example, aglycosylated biological or recombinantly derived molecule or aglycosylated enzyme, to form a larger complex. When used in accordancewith the present invention, one or more lectins are selected based uponsuch lectins' affinity for a specific glycan structure or a glycosylatedenzyme. Preferably, the lectin is selected for its biological affinityfor a specific glycan structure, or for a targeted glycosylated enzymeof interest whose presence is suspected in a test sample. In a preferredembodiment the methods and kits of the present invention contemplate theselection of lectins which are capable of recognizing anddiscriminatorily binding to specific glycoforms of an enzyme. Forexample, if the enzyme of interest in a selected test sample is aglycosylated form of the enzyme agalsidase alpha, then a lectin withaffinity for that enzyme (such as, for example, the lectin concanavalinA) would be preferentially incorporated into the assays, kits andmethods of the present invention.

The lectin's biological affinity for a specific glycan structure can beexploited in accordance with the present invention to isolateglycosylated enzymes or specific glycoforms of an enzyme in a testsample. Numerous lectins are commercially available, and information onthe binding specificity of a given lectin can be obtained from themanufacturer or as is described herein. Representative lectins include,but are not limited to, concanavalin A (Con A), wheat germ agglutinin(WGA), Jacalin, lentil lectin (LCA), peanut lectin (PNA). Lens culinarisagglutinin (LCA), Griffonia (Bandeiraea) simplicifolia lectin II (GSLII)Aleuria aurantia Lectin (AAL), Hippeastrum hybrid lectin (HHL, AL),Sambucus nigra lectin (SNA), Maackia amurensis lectin II (MAL II), Ulexeuropaeus agglutinin I (UEA I), Lotus tetragonolobus lectin (LTL),Galanthus nivalis lectin (GNL), Euonymus europaeus lectin (EEL), Ricinuscommunis agglutinin I (RCA), or combinations thereof.

In an alternative embodiment of the present invention, the capture agentmay comprise one or more fusion proteins, wherein such fusion proteinspreferably comprise one or more binding domains which are capable ofrecognizing and binding to one or more specific saccharide orcarbohydrate moieties of a glycosylated enzyme. For example themannose-6-phosphate receptor (M6PR) is capable of bindingmannose-6-phophate (M6P). The M6PR is approximately 300 kDa and consistsof approximately 15 extracellular domains. In one embodiment of theinvention a fusion protein capture agent comprises one or more M6PRbinding domains (e.g., M6PR domains 1, 3, 5, 9 and/or 11) whichdemonstrate affinity for M6P and/or other glycoforms of interest.Preferably, the selected binding domain demonstrates high affinity forthe saccharide or carbohydrate moieties of interest (e.g., M6PR domains3 and 9 correspond to high affinity M6P binding sites). The recombinantfusion protein capture agents of the present invention may optionallycomprise one or more regions or tags to facilitate purification,isolation or fixation of the capture agent (e.g., fixation to a solidsupport). For example, in one embodiment of the present invention sixhistidine residues (6His) may be linked to a M6PR binding domain tofacilitate the purification, capture or fixation of the capture agent toa solid support (e.g., a nickel chelate-coated 96-well plate). Thefusion protein capture agents of the present invention may furthercomprise one or more linker or spacer groups. In one embodiment of thepresent invention, the capture agent comprises the fusion proteinM6PR(D9)6His which may be expressed, for example, in HT1080 cells andpurified using nickel chelate affinity chromatography.

The present invention contemplates that capture agents (e.g., lectins)may optionally be fixed onto any portion of a solid support (e.g., maybe attached to an interior portion of a porous solid support material).As used herein, the terms “fixed”, “affixed” and “fixing” mean bound,adhered to or immobilized, for example, physically and/or chemically. Asthe term specifically relates to a solid support and a capture agent,“fixed” or “affixed” mean that the capture agent remains bound to, orimmobilized on, a solid support despite being subjected to washconditions or conditions which may alter such physical or chemicalbonds. Fixing may be, for example, spontaneous or result from anadditional step. Exemplary methods of fixing include evaporation (forexample, removal of volatile solvent), cooling (for example, resultingin a phase change from liquid to solid, or viscosity thickening), andcuring (for example, polymerization and/or crosslinking). The presentinvention contemplates the use of derivatized lectins as capture agentsto enhance the ability to fix a lectin onto a solid support. Forexample, biotinylated lectins demonstrate an enhanced ability to affixonto a solid support coated with avidin or streptavidin, (e.g., a 96well plate, optionally coated with avidin or streptavidin) and the useof such derivatized lectins are contemplated by the present invention,(Thompson et al, 1989, Clin. Chim. Acta 180(3):277-84). Other usefulderivatives include, but are not limited to, labels, fluorescent probessuch as rhodamine, or FITC, radioactive labels, electroactive labels,affinity tags (e.g., 6His) that can conjugate with secondary labels,oligonucleotides for PCR amplification, such as green fluorescentprotein or luciferase, chromogenic peptides, and any combinationsthereof.

In one embodiment of the present invention an array of biotinylatedlectins of differing carbohydrate specificities is immobilized directlyonto wells of streptavidin coated 96-well microtiter plates asillustrated in FIG. 1. Test samples containing different preparations ofa given glycosylated enzyme (e.g., aryl sulfatase A (ARSA), agalsidasealfa, galactocerebrosidase (GalC) or heparin N-sulfatase (HNS)) areallowed to bind and unbound material removed by a wash step.

One aspect of the present invention involves the identification andselection of capture agents (e.g., lectins) that demonstrate an affinityfor a glycan structure of interest, or for specific glycoforms of arecombinantly prepared enzyme and a determination that such lectins aresuitable for binding to the specific enzyme glycoform of interest. Theparticular capture agent selected for use in the present invention isgenerally determined based on the ability of such capture agent to bindto a specific glycosylation structure, such as mannose-6-phosphate,fucose, sialic acid, galactose, or any other specific sugar. In somecases, selection of a capture agent is based on the glycosylation of theenzyme targeted for capture. In other cases, the binding agents areselected based on empirical determinations such as high throughputassays. For example, a capture agent can be bound to a solid support andthe ability of the capture agent to bind a desired fraction ofglycosylated enzyme(s) may be determined by using means known to one ofordinary skill in the art (e.g., an ELISA assay). A capture agent foruse in the methods, assays and kits of the present invention may then beselected based upon such capture agent's ability to bind the glycanstructure of interest or a particular glycoform of an enzyme in a testsample. A suitable capture agent for use in the present invention maythen be used to identify the presence or absence of a glycosylatedenzyme of interest in a test sample. For example, such capture agentsmay be used to determine the presence of a particular glycoform of arecombinant enzyme in a harvest test sample extracted from variousstages in the development process of that recombinant enzyme.

A determination of the binding affinity of a particular capture agentfor a glycosylated enzyme of interest may be performed by fixing a panelof labeled capture agents (e.g., biotinylated lectins) on a solidsupport (e.g., a 96 well plate, optionally coated with avidin orstreptavidin). The capture agent panel is then contacted with a testsample suspected of containing the glycosylated enzyme of interest. Inthe presence of the glycosylated enzyme of interest, such enzyme willbind to the capture agent panel and form a bound enzyme complex.

In general, the compositions of the present invention are prepared byattaching (e.g., covalently attaching) at least two (e.g., at leastthree, at least four, at least five or more) capture agents onto a solidsupport or to a molecule that is attached to a solid support (e.g.,avidin, streptavidin or a nickel chelate). One embodiment of the presentinvention contemplates the selection of multiple capture agents whichare prepared by physically mixing at least two (e.g., three, four, five,or more) distinct capture agents and that are subsequently fixed on oneor more solid supports. The amount of a specific capture agent that isselected may be based on the test sample concentration and theapproximate concentration of the target glycoenzyme of interest in thattest sample. In general, the amount of the capture agent fixed onto asolid support to be contacted by a test sample is in excess of at leastabout 50% (e.g., at least 75% or at least 100%) of the amount of theportion of the target glycosylated enzyme predicted to bind to thecapture agent. Alternatively, capture agents are immobilized on a solidsupport at various capture agent/solid support ratios or concentrations.The binding capacity of the capture agent/solid support composition maythen be determined, or the amount of a test sample that can be loadedwithout saturating the solid support may be determined. In general, itis desirable that the amount of capture agent fixed on the solid supportis at least two-fold in excess of the amount of glycosylated enzyme thatis to be bound (e.g., a ten-fold excess or a 100-fold excess).

The invention provides, in part, a capture agent (e.g., multi-captureagent) affinity kit for use in practicing the methods and assays of theinvention. The kits of the present invention may include, for example,at least two capture agents (e.g., lectins) bound to one or more solidsupports. Examples of such solid supports include, without limitation,one or more beads or microbeads composed of silica, agarose, or apolymer, a plate (e.g., a microtiter plate), a slide (e.g., glass orpolymer slide), a nanowell plate, or polyethylene glycol or othersoluble polymer that can be precipitated or isolated by some otherphysical process to which a capture agent is bound. The capture agentsused in the invention can be attached to the solid support directly orindirectly (e.g., using an antibody or biotin) using methods known tothose of ordinary skill in the art, (e.g., using aldehyde functionalizedresins or linkers such as cyanogen bromide, carbonyl diimidazoleglutaraldehyde, epoxy, periodate, or bisoxirane) (Harris et al, 1989, InProtein Purification Methods. A Practical Approach, IRL Press, New York,N.Y.). In the case of particulate supports such as agarose beads, amixture of capture agents (e.g., lectins) may be fixed onto a singlebead, or in certain embodiments, a single type of capture agent isattached to each bead and the mixture of capture agents used in thecomposition is prepared by mixing at least two of these beadpopulations. Alternatively the capture agent may be attached to arestricted access media for the purposes of selecting glycosylatedenzymes of different molecular weight ranges.

The present invention further contemplates the binding of glycosylatedenzymes present in a selected test sample, (e.g., recombinant enzymes)to the capture agent to produce a bound enzyme. As used herein, aglycosylated enzyme is said to have “bound” its respective capture agentwhen it has associated with the capture agent through a non-randomchemical or physical interaction. The terms “bind” or “bound” mean thecoupling of one molecule (e.g., a glycosylated enzyme, such as theenzymes arylsulfatase A, agalsidase alfa and galactocerebrosidase) toanother molecule (e.g., a lectin, such as the lectins concanavalin A,sambucus nigra agglutinin and wheat germ agglutinin). Binding of anenzyme to a capture agent is preferably achieved under conditionssuitable for such binding to occur. Binding may be achieved by chemicalmeans (e.g., covalent or non-covalent in nature); however in a preferredembodiment, binding of the capture agent to the glycosylated enzyme ofinterest in the test sample is achieved by way of a covalent bond. Suchbinding need not be covalent or permanent. Following contact of thecapture agent with the test sample, the test sample is preferablycontacted with a wash solution such that the excess or unbound testsample fraction can be separated or removed, and if appropriatecollected for analysis.

Generally, the specificity in the detection of the bound enzyme will beperformed by determining the enzyme's activity via addition ofappropriate substrate and assay conditions as demonstrated in FIG. 1.The signal determined for a given capture agent (e.g., a lectin) coupledwith the known specificity of that capture agent will allow for a fast,high throughput, semi-quantitative structure assessment of theglycoforms present in the test sample. To determine the presence of theglycosylated enzyme of interest in a test sample, the bound enzymefraction is contacted with a substrate. As used herein, the term“contact” means that two or more substances (e.g., a bound enzyme and asubstrate) are sufficiently close to each other such that the two ormore substances interact or react (e.g., chemically or biologically)with one another. The term “substrate” refers to a molecule, complex,material, substance or reactant with which an enzyme reacts (e.g.,chemically or biologically), acts or binds. In particular, thesubstrates of the present invention may demonstrate a physiological,biological and/or chemical affinity for, or be able to be acted upon, bya corresponding enzyme. A substrate useful in the methods of theinvention can be native or modified. Modified substrates useful in theinvention retain the ability to be acted upon by the correspondingenzyme. Exemplary modifications suitable for substrates include, forexample, labeling to confirm the presence or absence of intrinsicenzymatic activity. One aspect of the present invention contemplates theselection of substrates based upon its ability to interact with, or bindto the enzyme of interest in a predictable and repeatable fashion. Forexample, a substrate with which an enzyme is known to react would bepreferable. Once a suitable substrate has been identified, thatsubstrate is preferably contacted with the bound enzyme and the presenceor absence of the anticipated reaction or interaction is assessed.

Based upon the known intrinsic enzymatic activity of the enzyme, in thepresence of the appropriate substrate the bound enzyme would be expectedto react, and accordingly signal the presence of that enzyme in the testsample. For example, the product of an enzyme reaction with a substrate(e.g., a molecular entity that is produced or liberated as a result ofenzyme acting on substrate) may provide a measurable signal indicativeof the presence of enzyme in the test sample, and that correlates withthe presence or amount of intrinsic enzymatic activity in the testsample. Alternatively, quantitative assessments of substrate binding ordepletion and/or assessments of the conversion of substrate to productmay be performed and used as a surrogate marker of intrinsic enzymaticactivity. Examples of suitable substrates for use in the presentinvention include 4-nitrophenyl-α-D-galactopyranoside,4-nitrophenyl-β-D-galactopyranoside and para-nitrocatechol sulfate.

Selection of the appropriate enzyme substrate and the subsequentdetermination of intrinsic enzymatic activity require an understandingof enzyme kinetics and in particular the catalytic properties of theenzyme(s) being evaluated. For example, enzymatic properties, such asMichaelis-Menton constants (K_(m)) and/or turnover numbers (K_(cat))relating to a particular substrate provide the basis for evaluating thesensitivity of an enzyme for one or more substrates and provideinformation regarding the reproducibility of the methods, kits andassays contemplated by the present inventions.

As used herein, the term “intrinsic enzymatic activity” refers to therepeatable reaction which an enzyme catalyzes or causes to occur, forexample in the presence of a substrate with which such enzyme is knownto react. In one embodiment of the present invention, the intrinsicenzymatic activity of an enzyme may be exploited to confirm the presenceor absence of such enzyme in a particular test sample. For example, manyenzymes have known and repeatable catalytic activity, which may beenhanced under certain conditions, such as the presence of theappropriate substrate. Intrinsic enzymatic activity may be measured byroutine means known to one of ordinary skill in the art (e.g.,colorimetric, spectrophotometric, fluorometric or chromatographicassays) by determining, for example the consumption or depletion ofsubstrate and/or the production of a product over time. In accordancewith the present invention, substrate depletion of about 5%, 10%, 20%,30%, 40%, 50% or more, or preferably about 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99% or more relative to the amount of substrateintroduced may be indicative of intrinsic enzymatic activity.Alternatively, following contacting an enzyme with a substrate, arelative increase in the formation of a product, or the conversion ofthat substrate to product, in each case of about 5%, 10%, 20%, 30%, 40%,50% or more, or preferably about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 99%, or more preferably 100% or more, may be indicative ofintrinsic enzymatic activity.

The methods described herein are useful for development of kits that canbe used for the detection of enzymes in a test sample. Such kits includeone or more capture agents (e.g., lectins or fusion proteins) fixed on asolid support which are capable of binding to a glycosylated enzymepresent in a selected test sample. The kits may also include additionalreagents such as buffers, substrates, enzymes, chemicals and othercompositions useful for further analysis and/or quantification of theligand-bound enzyme fraction. Kits can also include components for testsample preparation. The methods and kits described herein are useful forproviding a platform for the semi-quantitative assessment of thepresence of glycosylated enzymes in a test sample.

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate themethods, assays, kits and compositions of the invention and are notintended to limit the same.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents, Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification. Thepublications and other reference materials referenced herein to describethe background of the invention and to provide additional detailregarding its practice are hereby incorporated by reference.

EXAMPLE 1

Studies performed using the inventions disclosed herein havedemonstrated carbohydrate-specific binding of agalsidase alfa andgalactocerebrosidase drug substance material to several capture agents.The capture agents evaluated included the biotinylated lectins ConA(specific for core α-mannose structures), WGA (specific for dimers andtrimers of N-acetyl-glucosamine), and SNA (specific for Neu5Ac(α2,6)Galstructures).

Technical Feasibility

To determine the technical feasibility of the methods, assays andcompositions described herein, purified agalsidase alfa, aryl sulfataseA and galactocerebrosidase samples were initially assessed. The choiceof capture agent was initially limited to only a select few lectins withwell defined binding specificities. The lectin capture agents utilizedincluded: (1) concanavalin A (Con A), one of the most commonly usedlectins and known to bind avidly to core α-mannose structures ofN-linked high-mannose and biantennary complex-type oligosaccharides, (2)Sambucus nigra lectin (SNA) and Maackia amurensis lectin (MAL), whichrecognize Neu5Ac(α2, 6)Gal and Neu5Ac(α2, 3)Gal respectively, (3)Ricinus communis agglutinin I (RCAI), which binds terminal β1,4-linkedGal, and (4) wheat germ agglutinin (WGA), which binds poly-lactosaminerepeats Galβ1,4GlcNAc. Technical feasibility was based on sensitivity ofdetection. Feasibility was further evaluated using in-process testsamples provided from cell culture/process development streams.

Results

Method feasibility was evaluated using agalsidase alfa (Replagal®) drugsubstance and the biotinylated lectins ConA and WGA as the captureagents. The binding of decreasing agalsidase alfa concentrations (40ug/mL to 300 ng/mL) to the immobilized lectins was analyzed by measuringthe enzymatic activity of the bound enzyme. The enzyme activity ofagalsidase alfa was determined under steady-state conditions for thesynthetic colorimetric substrate 4-nitrophenyl-α-D-galactopyranoside.The substrate was hydrolyzed into 4-nitrophenol andα-D-galactopyranoside where the p-nitrophenol product was read at 405 nmonce the reaction was halted with an alkaline buffer. The binding curvesof agalsidase alfa (Replagal®) by both WGA and ConA (in absorbanceunits) are shown in FIG. 2.

The high avidity for ConA (which is specific for α-linked mannosestructures) at all concentrations tested demonstrated the highsensitivity of the assays and methods described herein. The binding forWGA (which is specific for dimers and trimers of N-acetyl-glucosamine)was less avid and followed a more classical titration curve. Thesestudies demonstrated the potential of the methods and assays of thepresent invention in terms of their sensitivity and high-throughputnature.

EXAMPLE 2

Method feasibility was further evaluated with galactocerebrosidase(GalC) drug substance material and the biotinylated lectins concanavalinA (Con A), wheat germ agglutinin (WGA), and Sambucus nigra lectin (SNA).The binding curves in FIG. 3 demonstrate both strong and selectivebinding to GalC.

To determine whether the methods and assays of the present inventionwere capable of detecting differences in the amount of GalC-associatedsialic acid, GalC samples were subjected to increasing amounts ofsialidase pre-treatment and then evaluated for binding. The resultsprovided in FIG. 4 demonstrate that controlled removal of sialic acidresults in reduced binding, providing evidence that the assays andmethods of the present invention are capable of measuring differences inthe amount of GalC-associated sialic acid.

EXAMPLE 3

To confirm the sialic acid binding specificity of Sambucus nigra lectin(SNA), aryl sulfatase A drug substance samples produced in CHO and humancells containing approximately 1 mol of sialic acid per mol of proteinin either α2,6-linkage (human cell-derived) or α2,3-linkage (CHOcell-derived) were analyzed for binding.

The binding curves provided in FIG. 5 illustrate both the selectivebinding for sialic acid in the α2,6-linkage and the α2,3-linkage.

EXAMPLE 4

To determine whether the assays, methods and compositions describedherein could be applied to actual harvest samples, early, middle, andlate galactocerebrosidase (GalC) harvest samples (H2, H10, and H17) froman early stage in the development process were analyzed for binding toSambucus nigra lectin (SNA).

The results shown in FIG. 6 demonstrate no difference in sialic acidbinding across all 3 harvests and importantly validate the intendedpurpose of the present invention.

EXAMPLE 5

The feasibility of the present invention was also evaluated with arylsulfatase A drug substance material derived from two differentproduction methods and a recombinant fusion protein which was preparedto function as the capture agent. The recombinant fusion proteinconsisted of the high affinity binding domain (D9) of themannose-6-phosphate receptor (M6PR) linked to six histidine residues(6His) to facilitate fixation of the M6PR to a nickel chelate coatedsolid support. The recombinant fusion protein construct is referred toherein as M6PR(D9)6His and was expressed in HT1080 cells, purified usingnickel chelate affinity chromatography, and was affixed onto a 96-wellplate.

To determine whether the methods and assays of the present inventionwere capable of detecting differences in the amount of aryl sulfatase Aassociated M6P associated with the two different production methods,aryl sulfatase A lots with known amounts of M6P were evaluated forbinding to immobilized M6PR(D9)6His (FIG. 7). Increasing concentrationsof test samples designated as either HGT-1110 or HGT-1111 (correspondingto the respective production methods) were added to the wells andallowed to bind for 2 hours at room temperature. The wells were washedin PBS and the enzyme activity was measured using the substratep-nitrocatechol sulfate.

As shown in FIG. 8, aryl sulfatase A from lot HGT-1111 which known toinclude more that 2× M6P per mol of protein as compared to arylsulfatase A from HOT-1111, displayed more avid binding as compared toaryl sulfatase A from lot HGT-1110. These results demonstrate that themethods, assays and kits of the present invention are capable ofmeasuring differences in the amount of aryl sulfatase A-associated M6P.

What is claimed is:
 1. A method for detecting the presence of an enzymein a test sample, wherein said method comprises the steps of: (a)contacting said test sample with at least one capture agent underconditions appropriate for binding of glycosylated enzyme in said testsample to said capture agent, wherein if said glycosylated enzyme ispresent in said test sample a bound enzyme is formed; (b) separatingsaid bound enzyme from said test sample; and (c) detecting intrinsicenzymatic activity of said bound enzyme, wherein the presence ofintrinsic enzymatic activity is indicative of the presence of enzyme insaid test sample, and wherein the capture agent comprises a fusionprotein.
 2. The method of claim 1, wherein said fusion protein comprisesat least one mannose-6-phosphate receptor (M6PR) binding domain.
 3. Themethod of claim 1, wherein said fusion protein comprises M6PR bindingdomain
 9. 4. The method of claim 1, wherein said fusion proteincomprises six consecutive histidine residues (6His).
 5. The method ofclaim 1, wherein said fusion protein is affixed on a solid support. 6.The method of claim 5, wherein said solid support comprises a metalchelate.
 7. The method of claim 5, wherein said solid support comprisesa multiple well microtiter plate.
 8. The method of claim 5, wherein saidsolid support comprises at least one bead.
 9. The method of claim 1,wherein the step of detecting intrinsic enzymatic activity of said boundenzyme is performed by contacting said bound enzyme with a substrate.10. The method of claim 9, wherein said substrate is reactive with anenzyme suspected of being present in said test sample.
 11. The method ofclaim 10, wherein said enzyme is agalsidase alfa and said substrate is4-nitrophenyl-α-D-galactopyranoside.
 12. The method of claim 10, whereinsaid enzyme is galactocerebrosidase and said substrate is4-nitrophenyl-β-D-galactopyranoside.
 13. The method of claim 10, whereinsaid enzyme is aryl sulfatase A and said substrate is p-nitrocatecholsulfate.
 14. The method of claim 10, wherein the step of detectingintrinsic enzymatic activity comprises quantitatively determining thedepletion of said substrate.
 15. A method for detecting the presence ofan enzyme in a test sample, wherein said method comprises the steps of:(a) contacting a test sample with at least one capture agent underconditions appropriate for binding of glycosylated enzyme, wherein ifsaid glycosylated enzyme is present in said test sample a bound enzymeis formed; (b) separating said bound enzyme from said test sample; (c)contacting said bound enzyme with at least one substrate; and (d)detecting the presence or absence of intrinsic enzymatic activity ofsaid bound enzyme, wherein the presence of intrinsic enzymatic activityis indicative of the presence of enzyme in said test sample, and whereinthe capture agent comprises a fusion protein.
 16. A kit for detectingthe presence of glycosylated enzyme in a test sample, wherein said kitcomprises (a) at least one capture agent, wherein said capture agent iscapable of binding said glycosylated enzyme, and wherein the captureagent comprises a fusion protein, and (b) at least one substrate,wherein said substrate is reactive with said glycosylated enzyme. 17.The method of claim 15, wherein the fusion protein comprises at leastone M6PR binding domain.
 18. The method of claim 15, wherein the fusionprotein comprises M6PR binding domain
 9. 19. The method of claim 15,wherein the fusion protein comprises six consecutive histidine residues(6His).
 20. The method of claim 15, wherein the fusion protein isaffixed on a solid support.
 21. The method of claim 20, wherein thesolid support comprises a metal chelate.
 22. The method of claim 20,wherein the solid support comprises a multiple well microtiter plate.23. The method of claim 20, wherein the solid support comprises at leastone bead.
 24. The method of claim 15, wherein the substrate is reactivewith an enzyme suspected of being present in the test sample.
 25. Themethod of claim 24, wherein the enzyme is agalsidase alfa and thesubstrate is 4-nitrophenyl-α-D-galactopyranoside.
 26. The method ofclaim 24, wherein the enzyme is galactocerebrosidase and the substrateis 4-nitrophenyl-β-D-galactopyranoside.
 27. The method of claim 24,wherein the enzyme is aryl sulfatase A and the substrate isp-nitrocatechol sulfate.
 28. The method of claim 24, wherein the step ofdetecting intrinsic enzymatic activity comprises quantitativelydetermining the depletion of the substrate.
 29. The kit of claim 16,wherein the fusion protein comprises at least one M6PR binding domain.30. The kit of claim 16, wherein the fusion protein comprises M6PRbinding domain
 9. 31. The kit of claim 16, wherein the fusion proteincomprises six consecutive histidine residues (6His).
 32. The kit ofclaim 16, wherein the kit further comprises a solid support.
 33. The kitof claim 32, wherein the fusion protein is affixed onto the solidsupport.
 34. The kit of claim 32, wherein the solid support comprises ametal chelate.
 35. The kit of claim 32, wherein the solid supportcomprises a multiple well microtiter plate.
 36. The kit of claim 32,wherein the solid support comprises at least one bead.
 37. The kit ofclaim 16, wherein the glycosylated enzyme is selected from the groupconsisting of aryl sulfatase A, agalsidase alfa andgalactocerebrosidase.
 38. The kit of claim 16, wherein the substrate isselected from the group consisting of4-nitrophenyl-α-D-galactopyranoside, 4-nitrophenyl-β-D-galactopyranosideand para-nitrocatechol sulfate.
 39. The kit of claim 16, wherein theglycosylated enzyme is agalsidase alfa, and the substrate is4-nitrophenyl-α-D-galactopyranoside.
 40. The kit of claim 16, whereinthe glycosylated enzyme is galactocerebrosidase, and the substrate is4-nitrophenyl-β-D-galactopyranoside.
 41. The kit of claim 16, whereinthe glycosylated enzyme is aryl sulfatase A, and the substrate isp-nitrocatechol sulfate.
 42. The kit of claim 16, wherein the kitfurther comprises a component to separate the bound enzyme from the testsample.